This proposal builds on the 20 years of this R01 discovering naked and radiolabeled antibodies for the treatment of leukemia and translating them into the clinic. Here we have advanced our goals to a more difficult and intriguing target for a mAb: the intracellular oncoprotein Wilms' tumor 1 (WT1), which is selectively expressed in neoplastic cells of most leukemias, and many other cancer types. We, and others, had previously identified peptides derived from the WT1 protein that induce HLA-A0201-restricted cytotoxic CD8 T cells, capable of killing tumor cells via TCR recognition. We hypothesized that a mAb specific for the WT1 peptide/HLA-A2 complex (mimicking a TCR) would be a novel and useful tool to study the immunobiology of WT1 and possibly an effective therapeutic agent. We discovered a human, cytotoxic mAb (ESK1) directed to a WT1 peptide that is presented on cell surface HLA-A0201. We have proposed a new approach to the treatment of patients with cancers by targeting an otherwise un-druggable intracellular oncoprotein. The preclinical performance of the mAb we discovered, more than exceeded our expectations as a possible therapeutic lead drug, but we need to fully understand its mechanism of action and host/cellular resistance in order to exploit its activity in humans. This approach would also provide a proof of concept for other tumor-specific, intracellular targets. Here, in a collaboration of two complementary labs across the street from each other (Scheinberg and Ravetch), we seek to understand in great detail the mechanisms of action of this new agent and to use the antibody as a highly specific tool to probe some aspects of the relationship between antigen presentation and effector function. AIM 1: To determine the mechanisms of possible resistance to ESK action in current mouse models of human leukemia and solid tumors. ESK1 therapeutic activity in vitro and in vivo is Fc-dependent, and ADCC dominates this activity. We will explore how the therapeutic activity is evaded causing relapse including possible target cell, agent and host pharmacokinetic, and host effector cell causes. AIM 2: By use of a tagged WT1 peptide- biotin probe, to understand the importance of epitope site number on the function of ESK1 in vivo and in vitro. We ask: How can therapy be achieved with so few epitopes (our target cells display 0.1-1.0% of typical mAb epitopes)? Is there a floor to this epitope number? Based on our preliminary data, we hypothesize that the number of mAb binding events required to recruit immune effector cells for ADCC (now unknown) will be far lower (<100/cell) than previously expected. If this is true, it would have profound implications for antibody specificity, activity ad therapy. We will also explore the minimal floor of epitope sites needed for therapy with an alpha emitting form (which requires only 1 hit to kill). AIM 3: To create a new, humanized Fc?R mouse as a model of cancer and characterize within it, ESK1 therapeutic activity. Can we create a more relevant mouse model to test human mAb? This tool will be critical to many investigators for the preclinical design and study of naked human mAb therapies. Within the model, the key FcR bearing effector cells will be determined.